PMSlOlC vector

We described here the cloning of Evasin open-reading frames into the pMSlOlC vector, the introduction of alanine mutations into these sequences and the screening of alanine mutants by phage ELISA and soluble ELISA (Figs. 1 and 2). 

Spread 100 pL of transformed cells on prewarmed 2YT plates containing 2% glucose and the appropriate antibiotic (ampiciUin for pMSlOlC vector). Incubate overnight at 30 °C. 

Transform chemically competent Machl bacteria. For each reaction, transfer 2 pL of Dpwl-treated DNA to 50 pL of bacteria. Incubate on ice for 30 min. Heat shock the cells for 30 s at 42 °C and immediately place them on ice for 1 min. Add 250 pL of room temperature SOC medium and incubate at 250 rpm for 1 h at 37 °C. Plate 80—100 pL of each transformation on prewarmed LB plates containing the appropriate antibiotic (ampiciUin for pMSlOlC vector). Incubate overnight at 37 °C. 

Incubate the ten tubes containing the cells at 250 rpm for 1 h at 37 °C. Pool the cells, measure the total volume of cells (ca. 20 mL) and spread onto five prewarmed 24 x 24 cm 2YT plates containing 2% glucose and the appropriate selection antibiotic (ampiciUin for pMSlOlC vector). Keep 200 pL of cells for the titration. Incubate overnight at 30 °C. Prepare a serial dilution of bacteria to titrate the number of colonies obtained and determine the size of the library. 

Digest the PCR product and the destination vector pMSlOlC with the B55HII and Sall-HF restriction enzymes. For each digestion (PCR product or pMSlOlC), prepare the following solution ... 

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